Plant Phosphatidylinositol 3-Kinase
Yuree Lee, Teun Munnik, and Youngsook Lee
Abstract Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position
of phosphoinositides. In Arabidopsis, only one PI3K exists, which belongs to
the class-III PI3K subfamily which makes phosphatidylinositol 3-phosphate
(PtdIns3P). The single AtPI3K gene is essential for survival, since loss of its
expression results in lethality. Although not much is known about the molecular
mechanism of its function, recent studies show that plant PI3K is important for
development and signaling, similar to yeast and animal systems. This includes
involvement in endocytosis, reactive oxygen species (ROS) production, and transcriptional activity. Many more interesting stories about the role of this enzyme in
the core of cellular activities of plants will be unfold as refined technologies are
applied to study this important enzyme.
1 Introduction
The phosphatidylinositol 3-kinase (PI3K) family of enzyme is the central player in
cell cycle regulation, signaling, and development in animal systems and thus has
been studied extensively (reviewed in Garcı´a et al. 2006). In plants, PI3K is also
important for development and signaling (Welters et al. 1994; Jung et al. 2002; Park
et al. 2003; Joo et al. 2005; Lee et al. 2008a, 2008b), though it has not been studied
as much as in animals. This is mainly because plants without the enzyme cannot
survive, and even reduction of expression of the enzyme results in severe retardation in growth and development (Welters et al. 1994). In addition, the low
quantity of 30 -phosphorylated inositol lipids makes biochemical detection very
difficult. Using diverse methods to overcome these problems, new aspects on the
Y. Lee (*)
POSTECH-UZH Cooperative Laboratory, Division of Molecular Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea
e-mail: [email protected]
T. Munnik (ed.), Lipid Signaling in Plants, Plant Cell Monographs 16,
DOI 10.1007/978-3-642-03873-0_6, # Springer-Verlag Berlin Heidelberg 2010
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role of PI3K have been found. Here, we briefly review studies on plant PI3K, with
the emphasis on its role in signal transduction and vesicle trafficking.
2 Molecular Classification of PI3K
PI3K phosphorylates the D-3 position of inositol phospholipids. Three different
classes can be distinguished, based on sequence homology and in vitro substrate
specificity (Wymann and Pirola 1998). Class-I PI3Ks are heterodimers composed
of a regulatory subunit and a PI3K catalytic subunit. They are involved in diverse
cellular phenomena, such as control of growth (Leevers et al. 1996), regulation of
cell cycle progression (Klippel et al. 1998; Gille and Downward 1999), DNA
synthesis (Roche et al. 1994; Vanhaesebroeck et al. 1999), cell survival (Yao and
Cooper 1995), actin rearrangements (Servant et al. 2000), and Ca2+ channel trafficking (Viard et al. 2004), by generating the phospholipid second messengers,
phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3], and PtdIns(3,4)P2 in the
plasma membrane of target cells.
Class-II PI3Ks are structurally distinct from the class I PI3Ks, and use only
phosphatidylinositol and phosphatidylinositol-4-phosphate as substrates. They are
constitutively associated with membrane structures (including plasma and intracellular membranes) and with nuclei. Several lines of evidence suggest a potential role
for these enzymes in agonist-mediated signal transduction (Foster et al. 2003),
migration of cancer cells (Maffucci et al. 2005), suppression of apoptotic cell
death (Kang et al. 2005), exocytosis (Meunier et al. 2005), pattern formation
(MacDougall et al. 2004), cytoskeletal organization (Katso et al. 2006), and insulin
signaling (Falasca et al. 2007).
Class-III PI3Ks use only phosphatidylinositol as a substrate, producing
PtdIns3P. The prototype for this enzyme, Vps34p, was first identified in Saccharomyces cerevisiae, where it is required for delivery of soluble proteins to the vacuole
(Herman et al. 1992; Schu et al. 1993). Subsequently, a human homolog was
identified, and meanwhile Vps34p-related PI3Ks are known to exist in a wide
range of eukaryotes, including Dictyostelium (Zhou et al. 1995) and Drosophila
(Linassier et al. 1997), and it is this isoform that is found in plants too (Hong and
Verma 1994; Welters et al. 1994; Molendijk and Irvine 1998). Since plants lack the
class-I and -II PI3Ks, differences between plant and animal PI3K signaling can be
expected.
3 Processes in Plants that Require Normal PI3K Activity
Pharmacological studies using the PI3K inhibitors, Wortmannin (WM) or
LY294002 (LY), have implicated a role for PI3K in various physiological events.
These include auxin-induced gravitropism (Joo et al. 2005; Jaillais et al. 2006), the
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formation of infection threads in Medicago truncatula roots inoculated with
Sinorhizobium meliloti (Peleg-Grossman et al. 2007), the salt-tolerance response
in Arabidopsis roots (Leshem et al. 2007), ABA-induced stomatal closure (Jung
et al. 2002; Park et al. 2003), actin reorganization (Choi et al. 2008), and tip growth
in root hairs (Lee et al. 2008a). PI3K is thought to modulate these processes by
regulating endocytosis and reactive oxygen species (ROS) production. Another
potential mechanism of plant PI3K action is via modulation of transcriptional
activity (Bunney et al. 2000).
Molecular genetic evidence suggests that PI3K is crucial for plant development,
both in vegetative and reproductive organs. Using antisense to reduce PI3K expression was found to impair leaf and stem development (Welters et al. 1994), while
a T-DNA insertion KO mutant is lethal and impaired in pollen development
(Lee et al. 2008b).
3.1
Roles of PI3K in Endocytosis and Protein Trafficking
In vivo, PtdIns3P can be tracked using a genetically encoded biosensor, which is a
fusion between GFP (or any other color) and two FYVE (from Fab1, YOTB, Vac1
and EEA1) domains in tandem which specifically bind PtdIns3P (Gillooly et al.
2000). Stably expressing lines of Arabidopsis plants and suspension-cultured
tobacco BY2 cells revealed strong colocalization with the late endosomal/prevacuolar
marker, AtRABF2b, and was found to partially colocalize with the endosomal tracer
FM4-64 (Voigt et al. 2005; Vermeer et al. 2006; see chapter, “Imaging lipids in
living plants”).
PI3K seems to play a role at different stages of vesicular trafficking, depending
on the cell type, as PI3K inhibitors have been found to suppress the initial uptake of
FM4-64 in tobacco cells and Arabidopsis roots under salt stress (Emans et al. 2002;
Leshem et al. 2007), the endocytic recycling of endosomes to the plasma membrane
in tobacco pollen tubes (Helling et al. 2006), and the fusion of late endosomes with
the tonoplast (Lee et al. 2008a).
PI3K-related endocytic routes have been suggested to deliver molecules important for plant signal transduction. For example, diacylglycerol (DAG), generated
from PtdIns(4,5)P2 (and/or PtdIns4P) via PI-PLC hydrolysis (see chapter, “The
Emerging Roles of Phospholipase C in Plant Growth and Development”) is delivered to a specific region of the plasma membrane in pollen tubes (Helling et al.
2006). Inhibition of PI3K disturbed the DAG localization pattern, as judged by the
accumulation of a DAG biosensor into YFPFYVE-labeled endocytic compartment,
with no or only weak accumulation at the plasma membrane. Based on these results,
Helling et al. (2006) suggested that DAG, generated at the flanks of the pollen-tube
tip, is internalized and reinserted into the plasma membrane at the apex via
PI3K-related endocytic routes. PI3K-related endocytic routes also deliver PIN
(auxin efflux transporters) proteins to specific regions of the plasma membrane.
Vesicular trafficking between the plasma membrane and endosomal compartments
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is necessary to maintain the polar distribution of PIN proteins (Geldner et al. 2001;
2003; Abas et al. 2006). This polar PIN localization is the primary factor determining
the direction of auxin flow in roots during the gravity response (Wisniewska et al.
2006). Inhibition of PI3K by wortmannin leads to the relocalization of PIN2 into
wortmannin-induced endosomal compartments, but did not affect PIN1 localization,
suggesting a specific role of PI3K in PIN2 cycling (Jaillais et al. 2006).
PI3K is essential for normal trafficking of proteins to and from vacuoles. PI3K
inhibitor interferes with targeting of vacuolar proteins in tobacco suspension cells
(Matsuoka et al. 1995). Reduction of free PtdIns3P level by expression of PtdIns3Pbinding protein interferes with vacuolar protein targeting in Arabidopsis protoplasts
(Kim et al. 2001). Moreover, PI3K inhibitors cause swelling or vacuolation of the
prevacuolar compartment (Tse et al. 2004) and block retrograde transport of
vacuolar sorting receptors to the TGN (daSilva et al. 2005; Oliviusson et al.
2006). Vesicular trafficking mediated by PI3K may rely on dynamic changes in
the actin cytoskeleton, since profilin, a regulator of actin dynamics, binds PI3K
in phosphorylation-dependent manner in Phaseolus vulgaris (Aparicio-Fabre et al.
2006).
3.2
Roles of PI3K in ROS Generation and ROS-Mediated
Signaling
ROS production is reduced by PI3K inhibitors in various cell types of plants
including root hair, guard cell, and pollen tube (Foreman et al. 2003; Park et al.
2003; Kwak et al. 2003; Potocky´ et al. 2007). This effect of the inhibitors is likely
due to the inhibition of PI3K-mediated activation/delivery of NADPH oxidase
(NOX), a major source of ROS. Based on the function of PI3K in endosomal
trafficking, there are three possible mechanisms by which PI3K could modulate
ROS production (Fig. 1): (1) by affecting the activity or distribution of plasmamembrane localized NOX, (2) by transferring exogenously produced ROS into
cytoplasm, (3) by regulating NOX activity in the endosomes. The first and the
second hypotheses are based on NOX localization at the plasma membrane and
ROS being produced at the apoplast, whereas the third one suggests that ROS is
produced inside endosomes. Two recent papers suggest that PI3K-dependent
plasma membrane internalization is linked to ROS production. Leshem et al.
(2007) reported that salt stress triggers PI3K-dependent plasma membrane internalization and ROS production within endosomes of root cells. Intracellular ROS
were encapsulated by endosomal membrane in root cells, and were interpreted as
the product of NOX internalized from the plasma membrane in response to salt
stress. In root hair cells, Lee et al. (2008a) also showed ROS inside endosomes and
the level of ROS in these organelles was reduced after treatment with LY.
In animal cells, PtdIns3P stimulates endosomal ROS generation through binding
the PX domain of p40phox, a soluble factor of the NOX complex (Ellson et al. 2006).
More and more endocytic organelles are considered as intracellular signaling
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Fig. 1 Diagram depicting three possible mechanisms of modulation of ROS generation by PI3K in
root hair system. First, PI3K-related endocytic recycling route can affect activity or distribution of
plasma membrane localized NADPH Oxidase (NOX), which produce ROS outside of cells (1).
Second, PI3K can affect import of exogenously produced ROS into cytosol. Diffusion of ROS
across lipid layers is very low and endocytosis mediated by PtdIns3P may contribute to transfer of
ROS into the cell (2). Finally, NOX can be recruited to the endosomes, where PtdIns3P is
localized, and produce ROS inside endosomes (3)
stations, where downstream cascades are activated after receptor–ligand complexes
are internalized into the endosomal compartment (Miaczynska et al. 2004), and
endosomal ROS plays a key role in regulating their activity (Li et al. 2006). In
plants, neither cytosolic factors of the NOX complex, nor intramembrane ROS
generation mediated by NOX has been shown. However, activated forms of receptors, e.g., the LRR receptors, FLAGELLIN SENSITIVE2 (FLS2), and the steroid
receptor kinase BRI1, have been observed to accumulate in endosomes (Robatzek
et al. 2006; Geldner et al. 2007). Whether and how endosome-localized FLS2 and
BRI1 activate downstream signaling cascades is unknown, but these results show
the potential of PI3K and endosomes as plant signaling components.
3.3
Roles of PI3K in Nucleus
Involvement of PI3K in nuclear function is based on the observation that PI3Ks are
associated with active nuclear transcription sites in plants (Bunney et al. 2000). A
catalytically active PI3K was demonstrated in isolated, detergent-resistant plant
nuclei and a monoclonal antibody raised against a truncated form of the soybean
PI3K was located at, or near, active transcription sites, both in the nucleolus and in
the nucleoplasm. The presence of PI3K and its product PtdIns3P in the nucleus is
not unique to plants. Nuclear PtdIns3P has been reported in BHK cells, human
fibroblasts, and HL-60 cells (Gillooly et al. 2000; Visnjic et al. 2003). In HL-60
cells, PtdIns3P level increases at G2/M phase of the cell cycle (Visnjic et al. 2003),
suggesting a role of the lipid in cell cycle. Although there are no reports yet for a
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link of PI3K with transcriptional regulation in animal cells, class I PI3Ks of animals
have been reported as important factors in various steps of cell division, such as
´ lvarez et al. 2003), regulation of cyclin/Cdk (Olson et al.
control of cell cycle entry (A
´ lvarez et al. 2001).
1995; Klippel et al. 1998), and progression of G2/M phases (A
Distinct features of FYVE containing proteins of plants also provide some clues
to the possible roles of PI3K in nucleus. Among the 16 proteins having FYVE
domain in Arabidopsis, nine contain tandem repeats of regulator of chromosome
condensation-1 (RCC1)-like domain (Van Leeuwen et al., 2004). RCC1 is a protein
that contains seven tandem repeats of a domain of about 50–60 amino acids and
functions as a nucleotide exchange factor for the nuclear Ran G-protein (Bischoff
and Ponstingl 1991). It regulates diverse biological processes including G1/S phase
transition (Matsumoto and Beach 1991), mating (Clark and Sprague 1989), the
processing and export of mRNAs (Kadowaki et al. 1993), and chromatin condensation (Sazer and Nurse 1994) in various eukaryotes. Although RCC1 homologs
have not been reported from plants, the RCC1 domain is found in many plant
proteins. Some of these may function similarly as RCC1 in the nucleus as suggested
from the in vitro assay using purified GST-RCC1 domain of PRAF1 in Arabidopsis,
which demonstrated the guanine nucleotide exchange of a Rab small GTPase
(Jensen et al. 2001). Further studies are required to understand whether plant FYVE
proteins with a RCC1-like domain function similar to RCC1 proteins in animals.
3.4
Roles of PI3K in Growth and Development of Plants
The broad and significant role of PI3K in plant growth and development was first
suggested by the results of Welters et al. (1994), who regenerated Arabidopsis
plants from calli transformed with an antisense construct of AtVPS34. Regeneration
of shoot and root was slow, flowers were formed, but the seed-set was poor. The
next generation of plants could not survive in kanamycin-containing medium. Even
in normal medium without antibiotics, leaves were abnormal in shape, and petiole
elongation and stem formation were impaired. In soybean, a PI3K is induced during
nodule development when membrane proliferation is required to establish the
peribacteroid membrane (Hong and Verma 1994).
In addition to the role of PI3K in vegetative tissue development, the enzyme also
plays a role in reproductive tissues (Lee et al. 2008b). When VPS34/vps34 heterozygous plants, harboring a T-DNA insertion, were self-fertilized, a segregation ratio
of 1:1:0 for wild type, heterozygous-, and homozygous mutant plants, respectively,
was obtained, thus homozygous mutants without PI3K expression were lacking.
These results suggested a gametophytic defect, which was further supported by
reciprocal crosses between heterozygous and wild-type plants. There was no transmission of the T-DNA insertion allele through the male gametophyte, indicating an
important role for PI3K during male gametophyte development. Male gametophytes of the heterozygous mutant plants showed reduced number of nuclei,
enlarged vacuoles, and reduced germination rate more often than the wild type.
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Is PI3K also required for female gametophyte development? Considering its
basic functions in cellular trafficking and nuclear division, it seems likely that PI3K
is also important for the development of female gametophyte, especially because it
involves many rounds of cell division. Consistent with this explanation, plants
expressing the AtVPS34-antisense construct were severely reduced in seed-set
(Welters et al. 1994), which suggested a role of the PI3K in development and/or
function of female reproductive organ as well. But how can we explain then the
results from the reciprocal crosses, which suggested that female allele of the pi3k
knockout is transmitted normally? A potential explanation for this discrepancy is
that a sufficient quantity of sporophytic gene product persists to complete megagametogenesis. The PI3K enzyme from previous generation may provide the lipid
during development of female gametophyte; female gametophyte inherits more
PI3K from the cytosol of previous generation than male gametophyte, is able to
complete its development normally. Such an explanation is consistent with the
observation that only the later steps in the mutant male gamete development were
defective, while the early steps of the process was normal.
4 Signal Transduction Pathway Activated at Downstream
of PI3K
The remarkably diverse and potent effect of PI3K-mediated signal transduction in
animal cells depends on the interaction of the lipid products of the kinases with
multiple protein partners. Signaling molecules related to class I PI3K of animal
cells have been identified and include phosphatidylinositol 3-phosphatase (PTEN),
3-phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B
(PKB)/c-Akt. PTEN is a lipid phosphatase which hydrolyzes the phosphate from
D3-position of inositol phospholipids, thus attenuating PI3K-mediated signaling.
AKT1 is recruited to the plasma membrane by binding PtdIns(3,4,5)P3 which is
produced by activated class I PI3K and is phosphorylated by PDK1. Phosphorylated
AKT1, in turn, phosphorylates numerous target proteins and thereby induces
multifaceted effects of PI3K. Identification of plant homologs of mammalian
downstream molecules of PI3K can be one way to obtain further clues about
plant PI3K signaling. Indeed, homologues of PTEN and PDK1 have been identified
in plants (Deak et al. 1999; Gupta et al. 2002). AtPTEN1 was shown to have
phosphatase activity against PtdIns(3,4,5)P3 and to play an important role in pollen
maturation after mitosis (Gupta et al. 2002). AtPDK1 has been shown to complement a yeast mutant lacking PDK1, to activate mammalian PKB in vitro, and to
bind a broad range of lipids, including PA, PtdIns3P, PtdIns(3,4)P2, PtdIns(4,5)P2,
and PtdIns(3,4,5)P3 (Deak et al. 1999). Further analysis of AtPDK1 revealed its
substrates, AGC2-1 kinase (OXI1), PINOID, and S6 kinase (Anthony et al. 2004;
Otterhag et al. 2006; Zegzouti et al. 2006) and its capacity to be regulated by PA
and PtdIns(4,5)P2 (Anthony et al. 2004).
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Although the conservation of AtPTEN1 and AtPDK1 indicate that PI3K-related
signaling is well conserved, plants lack the class-I PI3K and its product, PtdIns
(3,4,5)P3 (Meijer and Munnik 2003; Munnik and Testerink 2009). Thus, plant cells
may differ from animal cells in the downstream pathways. But if AtPTEN1 is
indeed the plant ortholog of animal PTEN, then what is its substrate(s)? Plants do
contain PtdIns3P and PtdIns(3,5)P2 as 3-phosphorylated phosphoinositides (Meijer
et al., 1999; Munnik and Testerink 2009), which may function as substrates of
PTEN. Similarly, if the AtPDK1 is, indeed, the plant ortholog of animal PDK1, the
immediate question to be resolved is whether any of the 3-phosphorylated phosphoinositides provides a specific site for recruitment of AtPDK1.
Signaling targets related to class-III PI3K of animal and yeast cells are proteins
that contain FYVE-, PH-, or PX-domains. Plants contain several of such proteins
(Van Leeuwen et al. 2004) and some of them have even been shown to bind
PtdIns3P (Deak et al. 1999; Jensen et al. 2001; Heras and Drøbak 2002; Vermeer
et al. 2006), although none have been characterized functionally in depth. Interestingly, Arabidopsis contains three proteins with a putative PX domain which are
members of the sorting nexin-like (SNX) proteins which are involved in endosomal
trafficking in yeast and animals. Recently, AtSNX1 has been shown to play a role in
auxin-carrier trafficking which is sensitive to WM and was proposed to define a
sorting endosome (Jaillais et al. 2006, 2008). Clearly, more of these studies are
required to reveal the roles of proteins functioning downstream of PtdIns3P in
vesicular trafficking and protein targeting in plants.
5 Conclusion and Prospects
PI3K is emerging as important enzyme in plant signal transduction, regulating ROS
production and modulating the recycling of plasma membrane proteins and lipids. It
is also likely to be important for cell cycle regulation, via its role in nuclear division
and transcriptional control. To better understand the whole picture of PI3Kmediated pathways, downstream effector molecules have to be identified. In addition, improved genetic analyses are required, using conditional mutations driven by
specific promoters. The cell biology of PtdIns3P dynamic and its targets may also
provide further information on the role of PI3K in signaling and protein trafficking.
With all these new tools, exciting times are ahead of us.
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